Whole-Exome Sequencing Analysis of Tumor and Non-Neoplastic Tissues

WES was performed on matched tumor and non-neoplastic tissues using previously described techniques.^{19 (link),20 (link),22 (link),23 (link)} DNA was extracted from macrodissected FFPE or fresh frozen tumor tissue along with peripheral blood lymphocytes or buccal tissue using the Promega Maxwell 16 MDx. The Agilent HaloPlex kit, an enzymatic capture method, was used for library preparation. Both neuropathology review and CLONET software (OMICS_07304), a computational tool to estimate ploidy and tumor content, estimated tumor content. An Illumina HiSeq 2500 system (2 × 100 bp) was used to sequence all samples. A total of 21 522 genes were analyzed with an average coverage of 90× and 94× for tumor and non-neoplastic specimens, respectively. All bioinformatics analyses were performed according to the Clinical Laboratory Improvement Amendments (CLIA)-approved Whole-Exome Sequencing Test For Cancer-EXaCT-1-pipeline version 09.^{20 (link)}Germline variants were screened based on our most recent germline pipeline.^{19 (link),20 (link)} For the purpose of the study, we included only variants in 152 cancer-associated germline genes. All candidate pathogenic germline variants detected were manually reviewed with the Integrative Genome Viewer.^{24 (link)}